Development of Methods for Measuring Protein C Inhibitor and Antithrombin: Use of Monoclonal Antibodies against the Reactive Center Loop-Inserted Forms of the Serpins

Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that are involved in the regulation of coagulation. Like other inhibitory serpins, PCI and AT adopt different structural conformations that are related to their functions. The cleaved, inactive form is a result of cleavage by a protease, and the latent form, which is also inactive, can arise due to a mutation in the serpin. Methods that quantify the different forms can be useful as diagnostic tools. The aim of this work was to develop such methods for measuring AT and for measuring cleaved PCI in complex with APC (APCI-PCI). The high-affinity monoclonal antibody M36 (KD ~50 pM) was produced against cleaved PCI. M36 is specific enough to discriminate between cleaved and native PCI and thus it was used as the catcher antibody, along with a monoclonal antibody against protein C as detector, in an immunoassay developed to measure the APC-PCI complex in blood plasma. The mean concentration of APC-PCI was found to be 0.30 µg/L in healthy individuals, and the levels of this complex were elevated in patients who had undergone hip surgery and were lowered in patients treated with warfarin. A crystal of cleaved PCI was obtained, which revealed a short helix A, and this is similar to the two hormone-binding serpins called corticosteroid-binding globulin and thyroxine-binding globulin. A potential binding site for retinoic acid was found in PCI, and moreover, possible novel binding sites for heparin were exposed, which suggested an alternative model for heparin-activated inhibition of APC by PCI. The monoclonal antibody M9 against reactive center loop-inserted antithrombin (AT) was produced and was found to have affinities (KD values) of 3.07 and 2.27 µM for latent and cleaved AT, respectively. An immunoassay for measuring cleaved AT was developed using M9 as catcher antibody and the monoclonal antibody M27 (specific for cleaved and native AT) as detector. The median level of cleaved AT was 1.3 mg/L in healthy individuals. No correlation was found between cleaved AT and the thrombin-antithrombin (TAT) complex in patients with deep venous thrombosis. The monoclonal antibody 8C8 was produced against latent AT, and it showed affinities (KD values) of 0.92 nM, 0.57 µM, and 5.65 µM for latent, native and cleaved AT, respectively. An immunoassay was developed for measuring latent AT, using M9 (specific for cleaved and latent AT) as catcher antibody and 8C8 as detector. The assay showed that the median level of latent AT was 4.7 mg/L in healthy individuals. Commercial preparations of native AT were found to contain ?10% latent AT. In addition, a weak interaction (KD = 51 µM) was observed between latent and native AT, which contradicts the formation of a stable dimer in vivo

An immunochemical method for quantitative determination of latent antithrombin, the reactive center loop-inserted uncleaved form of antithrombin.

Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be similar to 4.8 mg L-1 in healthy individuals. The K-D of the interaction between native and latent AT was found to be 51 mu M, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood

Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

beta-Microseminoprotein (MSP), a 10kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is

Structure of native protein C inhibitor provides insight into its multiple functions

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into beta-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions

N-glycans and the N terminus of protein C inhibitor affect the cofactor-enhanced rates of thrombin inhibition

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta 6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta 6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states

Efficacy and safety of controlled ovarian stimulation using GnRH antagonist protocols for emergency fertility preservation in young women with breast cancer-a prospective nationwide Swedish multicenter study

STUDY QUESTION: How efficacious and safe are the current approaches to controlled ovarian stimulation (COS) aimed at fertility preservation (FP) in women with breast cancer (BC)? SUMMARY ANSWER: In women with BC undergoing COS aiming at egg/embryo cryopreservation, letrozole-based protocols and those randomly started were equally effective compared with conventional COS, and the overall survival was similar between the women that proceeded to FP and those who did not. WHAT IS KNOWN ALREADY: Cryopreservation of oocytes and embryos is an established method for FP in women with BC. Recent improvements to COS protocols include concomitant use of letrozole, random-cycle start day of stimulation and the use of GnRHa for the egg maturation trigger. To date, limited sample size of the available studies has not allowed investigation of differences in the efficacy of the different approaches to COS for FP in this patient population. STUDY DESIGN, SIZE, DURATION: A prospective multicenter study with national coverage including 610 women with BC counseled between 1 January 1995 and 30 June 2017 at six Swedish FP regional programs. PARTICIPANTS/MATERIALS, SETTING, METHODS: After counseling, 401 women elected to undergo COS. Treatments differed in the use or not of concomitant letrozole, a conventional or random-cycle day COS initiation and the use of hCG versus GnRHa trigger for oocyte maturation. Numbers of cryopreserved oocytes and embryos were defined as primary outcome. Pregnancy attempts, reproductive outcomes and long-term survival, investigated by the linking of individuals of the cohort to the total population register of the Swedish Tax Agency (up to 25 November 2018), were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Using letrozole or not resulted in similar numbers of oocytes and embryos cryopreserved (mean(oocytes)=9.7 versus 10 and mean(embryos) 4.0 versus 5.3, respectively), similar to COS with random versus conventional start (mean(oocytes) 9.0 versus 10.6 and mean(embryos) 4.8 versus 4.8). In COS with letrozole, a GnRHa trigger was associated with a higher number of oocytes retrieved (P&amp;lt;0.05) and embryos cryopreserved (P&amp;lt;0.005), compared with conventional hCG trigger. Of 99 women who returned to fertility clinics after cancer treatment, 32 proceeded to thawing of oocytes or embryos and 10 of them had live births. The all-cause survival between the women that underwent COS and those who did not was similar and did not differ between the two groups. LIMITATIONS, REASONS FOR CAUTION: Data on tumor characteristics and estrogen receptor (ER) status were not known for all women at the time of FP counseling and planning of COS, thus protocols with letrozole have been used for both estrogen-sensitive and non-estrogen-sensitive BC. For the same reason, subsequent adjustment for ERs in the BC or tumor characteristics as potential confounders were not performed as these parameters were not available and did not influence the provision of FP through COS. WIDER IMPLICATIONS OF THE FINDINGS: The results of our study support the premise that recently introduced potential improvements to COS protocols for FP in women with BC are efficacious and safe.Funding Agencies|Swedish Cancer SocietySwedish Cancer Society; Stockholm County CouncilStockholm County Council; Percy Falk Stiftelsen; Radiumhemmets Forskningsfonder; Swedish Breast Cancer Association; Karolinska InstitutetKarolinska Institutet</p